7

Microbiology of Meats
DOUGLAS L. MARSHALL and M. FARID A. BALA Mississippi State University, Mississippi State, Mississippi

I. INTRODUCTION II. MEAT CONTAMINATION AND DECONTAMINATION III. MEAT AS A SUBSTRATE FOR MICROBIAL PROLIFERATION IV. MICROBIOLOGY OF FERMENTED AND CURED MEATS V. MEAT-ASSOCIATED FUNGI VI. MEAT-ASSOCIATED PARASITES VII. MICROBIAL MODELING VIII. SUMMARY ACKNOWLEDGMENTS REFERENCES

I. INTRODUCTION Contamination of sterile animal muscle used as food is a direct consequence of slaughtering and dressing of animal carcasses. Wide ranges of microorganisms from different sources are introduced onto moist muscle surfaces that are rich in nutrients. It is argued that only a small portion (10%) of these microorganisms is capable of survival and proliferation during storage, distribution, and retail sales of meats. Additionally, an even a smaller portion will eventually predominate and cause spoilage. Survival and proliferation of microorganisms deposited on meat surfaces depends on their ability to withstand processing and storage conditions and to utilize available nutrients in the muscle through assimilation or proteolysis of complex molecules into readily utilizable substrates. Over the years, efforts to preserve meats have focused on retarding microbial growth or killing selected contaminants by applying chemical, physical, or biological treatments whose net outcome should slow or prevent the growth of spoilers or allow harmless fermentative microorganisms to predominate. In either case, successful treatments extend product shelf life and allow its delivery from farm/processor to remote consumption areas.

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Microflora of meat products available to consumers at the retail level is a reflection of the environment in which they were processed and the conditions under which they were stored. From a numerical standpoint, using the per capita meat consumption of 88 kilograms (2,109) and an average microbial load of 10,000 colony forming units (CFU) per gram, a family of four annually brings into their home nearly 1 billion microorganisms associated with raw meat products. The nature and composition of viable microorganisms associated with consumer health risks and associated economic impact on the meat industry vary with the nature of the animal, processing, packaging, storage, and handling conditions. Meats can acquire a large variety of pathogenic and spoilage microorganisms during primary and further processing (Table 1). Pathogens can include Clostridium perfringens, Staphylococcus aureus, Salmonella spp., pathogenic Escherichia coli, Campylobacter spp., Yersinia enterocolitica, Listeria monocytogenes, and Aeromonas hydrophila (44,49,112). Meat pathogens can cause self-limiting human enteric diseases or systemic and fatal infections of the immunocompromised, the elderly, and the young. Spoilage of meats is largely dependent on initial microbiological quality and subsequent storage conditions. Pseudomonas spp. predominate in chilled airstored meats, (38) Enterobacteriaceae in temperature-abused meats, (83) lactic acid bacteria and Micrococcaceae in meats packaged with preservatives (82,88) and Brochothrix thermosphacta in vacuum- and modified atmospherepackaged products (118). Gill (42) reviewed the potential sources of meat contamination during slaughter and butchering of food animals. Animal health, hide, viscera, feces, oral microflora, and carcass handling are all potential sources of cross-contamination of sterile muscle during dressing operations. Ultimately, the microbiological quality of dressed carcasses relies a great deal on the skill level of operators during dressing operations, in particular skinning and evisceration, more so than on physical facilities or the types of stock slaughtered (87). Several decontamination approaches have been proposed to enhance the microbiological quality and safety of dressed carcasses (24). Treatments with organic acids (30), hot water (56), steam pasteurization, and steam carcass vacuuming (122) have been implemented in some processing plants. Bacterial species vary widely in their susceptibility to decontamination treatments, with 2 to 3 log population reductions possible (24,26). Determining the microbiological quality of dressed animal carcasses requires obtaining samples for laboratory analysis. Classical sampling methods have included excising a meat surface portion (16,80) or swabbing a defined surface area delimited by a template (57) to determine microbial loads per unit area. Obtained samples are either suspended and homogenized in a diluent and bacterial counts determined directly by spread plate, pour plates, hydrophobic grid membrane filtration (HGMF), or indirectly by ATP bioluminescence or impedance measurements (51,58). According to Gill and Jones (51), HGMF can enhance detection sensitivity to 1 CFU/100 cm2. For routine microbiological monitoring of hygienic quality of carcasses, USDA-FSIS mandates combining samples obtained from three sites of a given carcass and determining E. coli counts (127). In any event, carcass sampling remains a technique and its usefulness in predicting microbiological quality or microbial profiles depends on obtaining a true representative sample in high volume operations (27,79,119). From a practical standpoint, it is impossible to monitor dressed carcasses for all potential pathogens and spoilage microorganisms they may harbor. This consideration limits meat microbiological monitoring to either total aerobic plate counts or to E. coli counts as an indicator of fecal contamination (127). The latter approach is currently favored in view that total aerobic plate counts bear no indication to the potential presence of pathogens (117). However, the use of E. coli counts, or other potential indicator organisms, shows that no single indicator organism is effective for all types of foods (14).

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Consumers view quality aspects of meats as being good taste, tender, juicy, fresh, lean, healthy, and nutritious. However, cross-cultural differences exist in what is designated as fresh meat (60). The butcher in most developed countries may no longer be the guarantor of meat freshness, quality, and safety because supermarket chains supplied from major processors dominate the meat processing and retailing business. This chapter will address the microbiology of meat products with emphasis placed on the roles that processing, storage, and retailing exert on the microflora. II. MEAT CONTAMINATION AND DECONTAMINATION The microbiological profile of meat products presented to consumers is the sum total of slaughtered animal health, conditions under which it was reared, quality of slaughtering, processing, packaging, and conditions under which the meat was stored. Table 1 lists the common genera of microorganisms found on fresh, processed, and vacuum-packaged meats. Gill (42) reviewed the potential sources of meat contamination during slaughtering and butchering of food animals. Animal health, hide, viscera, feces, oral microflora, and carcass handling are all potential sources of cross contamination of sterile muscle during dressing operations. With cattle and sheep, the major source of initial meat contamination is the animal hide or fleece (6,7,42,62). These sources are exposed to soil, feces, water, and oral microorganisms during animal rearing. It would seem logical that cleaning the hide or fleece before dressing should reduce the number of potential contaminants; however, several studies have failed to confirm this point (9,10,128). On the other hand, scalding treatments applied to pigs destroys gram-negative bacteria, leaving predominantly gram-positive bacteria as survivors (39). Subsequent pig carcass dehairing reintroduces gram-negative bacteria from accumulated detritus and contaminated recirculated water (12,44,49). Animal hides not only introduce spoilage bacteria such as Pseudomonas, Acinetobacter, and Moraxella but also may introduce potential pathogens such as C. perfringens, S. aureus, Salmonella spp., E. coli, Campylobacter spp., Y. enterocolitica, L. monocytogenes, and A. hydrophila (44,49,112). Foodborne pathogens of animal origin can cause human gastroenteritis, and in high-risk populations they can lead to systemic and sometimes fatal infections (8). Other potential risks of meat contamination involve the potential transfer of antibiotic-resistant microorganisms to dressed meats and their ability to subsequently transfer this resistance to other microorganisms in the human gut (102). Evisceration is another critical step where operator skill is required to avoid spilling fecal matter onto skinned carcasses. It has been shown that bacteria deposited on carcasses during eviscerating operations predominantly originate from the mouth and the anus (50). Ultimately, the microbiological quality of dressed carcasses heavily relies on the skill level of operators during skinning and evisceration, more so than on processing plant physical facilities or the type of animal slaughtered (87,90). Little information exists on the impact of misprocessing events on microbiological quality of dressed carcasses. Gill (42) advocated treating common mishaps, such as spilling of gut contents onto carcasses or contamination introduced by operator handling, as events that necessitate special treatment. Accidentally contaminated carcasses should be flagged and detained for extra trimming and washing to remove visible contamination. Gill and Jones (50) showed that carcass pasteurization combined with a modified dressing process that prevents contamination from animal mouth and viscera can produce dressed pig carcasses with average E. coli counts of 1 per carcass and total counts of 2 CFU/cm2. Enhancing the microbiological quality of

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Table 1 Genera of Microorganisms Commonly Found on Meats

Type of meat Gram Rxn BACTERIA Achromobacter Acinetobacter Aeromonas Alcaligenes Alteromonas Bacillus Brochothrix Campylobacter Carnobacterium Citrobacter Clostridium Corynebacterium Enterobacter Enterococcus Escherichia Flavobacterium Hafnia Janthinobacterium Klebsiella Kluyvera Kocuria Kurthia Lactobacillus Lactococcus Leuconostoc Listeria Microbacterium Micrococcus Moraxella Paenibacillus Pantoea Pediococcus Proteus Providencia Pseudomonas Psychrobacter Salmonella Serratia Shewanella Staphylococcus Vibrio Weissella Yersinia Fresh X XX XX X X X X X X X X X X XX X X X X X X X X X X X X X XX X X X X X XX XX X X X X X X Processed Vacuum packaged

X X X X X

X X

XX XX

X X X

X X XX

X X

X XX X X X X X X X X

X X XX X X X

X X

X X X X

X X X X

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Table 1 Continued
Type of meat Gram Rxn YEASTS Candida Cryptococcus Debaryomyces Hansenula Pichia Rhodotorula Saccharomyces Torulopsis Trichosporon MOLDS Acremonium Alternaria Aspergillus Aureobasidium Botrytis Chrysosporium Cladosporium Fusarium Geotrichum Monascus Monilia Mucor Neurospora Penicillium Rhizopus Scopulariopsis Sporotrichum Thamnidium Fresh XX X X X X X XX X X X X X X XX X XX X X XX X X XX XX XX Processed X XX Vacuum packaged

X X

X XX X X X X X X XX X X X

X known to occur, XX most frequently isolated. Source: Dillon (25); Garcia-Lopez et al. (35); Jay (78).

dressed carcasses entails recognizing microbial hazards (12,91) and designing strategies to limit their spread to muscle foods during carcass dressing. Following dressing, carcasses are split and knife trimmed to remove visible contamination and bruised tissue (126). Studies of trimming practices have indicated that they are purely of aesthetic value and do not contribute to enhancing microbiological quality of dressed carcasses (42,43,57). Furthermore, trimming causes loss of meat and requires extra operator time and effort without necessarily improving microbiological quality (112). The requirement to meet pathogen reduction performance standards has prompted a number of studies in which antimicrobial intervention strategies have been compared. Washes with chemicals have been most advocated. Considerable literature exists on the antibacterial efficacy of dilute solutions of organic acids, hydrogen peroxide, chlorine, chlorine dioxide, and organic acid salts (63,93,108,110). Overall, such interventions can result in reduction of contaminants by 1 to 2 logs (24). In addition to benefiting meat safety, these

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Figure 1 Time required for spoilage development in frankfurters held at different refrigeration
temperatures. (Data from Ref. 130.)

interventions can also improve meat quality. For example, a reduction in the numbers of psychrotrophs by 4 logs can greatly increase the refrigerated shelf life of frankfurters (Fig. 1). Studies that have addressed meatborne pathogens have shown varying susceptibilities to such treatments. Campylobacter jejuni and Y. enterocolitica are among the most susceptible pathogens to acid treatment (92,94), E. coli O157:H7 and L. monocytogenes are more resistant (13,108,120). Smulders and Greer (123) reviewed other prospects of organic acid decontamination and resultant adverse effects on muscle foods. Using organic acids as a decontaminant in abattoirs has been recommended (127); however, a primary concern to meat exporters is that European Union countries do not allow for product decontamination treatments with anything other than potable water (123). The use of hot water to decontaminate dressed carcasses has been studied with beef (15,23), sheep (28), pork (56), and buffalo (113). A typical outcome of carcass washing was the reduction of microbial loads by 2 logs with hot water treatment (80C for 10 sec) (122). Another possible outcome of all wash operations is the uniform redistribution of contaminants from heavily soiled areas to the whole carcass (42). Hot water washes largely have not been adopted commercially in view that large volumes would be needed to uniformly heat a carcass surface. Moreover, economic reasons would impose recirculating hot treatment water, but sanitary concerns would not entertain such cost reductions (42). Alternatively, steam pasteurization has been advocated as a more viable option (19,29,97); however, to ensure adequate carcass surface heating, supra-atmospheric pressures are needed, which entails the need for specialized containment chambers (19). In commercial applications, steam is applied for 6.5 sec without appreciable sensory degradation and results in 2 log and 1 log reduction in E. coli and total aerobic counts, respectively (29,97,106). Steam pasteurization enriches growth of gram-positive bacteria on meats while reducing the more thermally susceptible gram-negative enteric pathogens (42). A less expensive alternative to steam pasteurization chambers involves the use of hot water or steam vacuum hand-held wands. Selected carcass areas can be treated by this method to remove visible contamination. A favorable report exists on the steam-vacuum approach to treat artificially contaminated meats (29). Use of this method in a commer-

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cial setting was advocated as an alternative to cleaning soiled carcass areas and to reduce the need for subsequent trimming (29). Following the dressing process, carcasses are transferred to chiller rooms. Rapid cooling before the development of rigor results in toughening of the muscle and subsequent loss of meat quality (125). On the other hand, slow cooling ensures rigor and allows for spoilage and pathogenic bacteria to proliferate (42). Commercial chilling of dressed carcasses use chill tunnels that rely on blasts of cold air (48) or spraying of carcasses with chilled water (55,59). It has been proposed that very fast carcass cooling can ensure muscle tenderness and microbiological quality (81); however, major capital spending would be required to implement fast cooling systems in abattoirs (42). Another consequence that accompanies air chilling is drying of the carcass surface by the reduction of surface moisture. Typically a 0.5 log microbial count reduction is observed as a consequence of surface drying (96). Alternative techniques have desiccated the carcass surface with dry heat (18). The major objection to carcass drying is the accompanying carcass weight loss (39). Accordingly, many abattoirs in North America have adopted intermittent spraying with water during the first hours of cooling to avoid weight loss (42). Gill (42) proposed an explanation for the observed reduction in Gram-negative and E. coli counts on spray-cooled carcasses. His twofold mechanism was that (a) spray cooling washed away surface bacterial loads and (b) surface freezing reduces gram-negative bacteria due to their greater susceptibility to freezing. Offal or organ meats are collected in bulk containers during carcass dressing. The geometry of their storage adversely affects subsequent chilling. Product on the periphery of storage containers cools faster than product in the center of the bulk container (42). Even in freezers of high cooling capacities, product at the centers of large containers will cool more slowly (5), thus providing greater opportunity for microbial proliferation. Offal meat pH is usually 6.0, which implies that the major hurdle in restraining microbial growth would be temperature and potential anaerobic conditions produced at the center of bulk containers (45,47). Enhancing the storage potential of offal can been achieved by vacuum or CO2 packaging to noticeably extend shelf life up to several weeks (46). However, efforts by the meat industry to ensure the microbiological quality of offal will remain proportional to their economic value on the market. Hot boning involves breaking down an animal carcass immediately after dressing as boxed manufacturing meats or for further processing into comminuted, cured, or cooked products. Boxing and stacking of hot boned meats in a cooler suffers similar limitations as the earlier described chilling of offal (111). However, temperature control is possible using ice or liquid carbon dioxide (42). Studies are needed to elucidate the effects of rigor development on the proliferation of microorganisms in hot-boned meats (42). Following chilling, carcasses are either broken down into primal cuts or transported to other processing plants for further processing. In either event, personnel hygiene and proper sanitation of equipment and work surfaces are prerequisites to ensure microbiological quality and safety of processed meat products. Visible cleanliness of equipment and work surfaces although advocated (127) may not be a true reflection of sanitary conditions unless coupled with microbiological or bioluminescent assays to ensure efficacy (34). Important sources of microbial contamination of processed meat products include detritus accumulated in inaccessible parts of equipment that subsequently contaminate products that come in touch with these surfaces (69,70). Under these circumstances, bacterial biofilms flourish due to repeated applications of water and the constant availability of nutrients from accumulated detritus. In cooled work environments, cold-tolerant (psychrotrophic) pathogens such as L. monocytogenes, Y. enterocolitica, and A. hydrophila can flourish

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(49,107). Ensuring the quality of processed meats entails making sure that biofilms do not persist day to day on work surfaces and equipment (131). Processed meats and primal cuts await their distribution in coolers. With raw meats, low storage temperature and low pH (5.5) due to rigor are the only two hurdles to slow bacterial proliferation. At all times, meat temperatures should not exceed 7C to prevent pathogenic mesophiles from proliferating (11). Consequently, storage and transport of meat at chilled temperatures lower than 7C is regarded as safe, although some pathogenic psychrotrophs can proliferate at this temperature (42,103). Limiting psychrotroph growth and further extending shelf life can be achieved by lowering the temperature of boxed meat to 1.5C without obvious ice formation (54). At temperatures of 0, 2, and 5C, chilled meat will have a storage life of 70%, 50%, and 30% of the storage life of meat stored at 1.5C (42). Alternatively, meats can be frozen for extended periods of time; however, the microbiological concern for frozen meats requires that temperatures be maintained below 5C, the minimum growth temperature of yeast and mold fungi (42,84,85). Although chill temperatures inhibit microbial growth, pathogens may survive for prolonged periods in meats even though they do not multiply. The microflora of meats available to consumers is the total sum of microorganisms acquired during processing of animal muscle food. Animal health, dressing skills, personnel hygiene, abattoir cleanliness, and adequate storage and holding temperature during distribution and retail influence the constitution and number of microorganisms present (17,72). It is unlikely that a single intervention can fully enhance quality and safety of meats (42). Rather, consumer education (1) coupled with an integrated approach, which ensures better understanding and optimization of each processing step (41), are more likely to enhance the future quality and safety of meats available to consumers. III. MEAT AS A SUBSTRATE FOR MICROBIAL PROLIFERATION Proliferation of microorganisms in meats is dependent on several factors, which include microflora composition, product temperature, previous product treatments, pH, available nutrients, oxidation-reduction potential, and the atmosphere surrounding the product. Many of these factors are not constant throughout the shelf life of a meat product. Understanding the influence of these factors on microbial growth and survival and the impact on meat spoilage has been greatly aided by the pioneering work of Ingram and Dainty (20,21,74,75). More recently, Nychas et al. (101) reviewed the subject of chemical changes associated with stored meats. Common defects of meats and associated bacteria are shown in Table 2. Microbial proliferation in meats occurs in the aqueous phase surrounding the product. This phase is rich in substrates readily utilizable by almost all microorganisms (98). Frozen (12C) meats prevent the growth of contaminating microorganisms but allow for their abundant survival during storage. Spoilage of thawed meats is due to the number and type of microbes present before freezing and the time/temperature conditions of the product during thawing. Thawed meats are often more perishable than fresh meats because of the abundance of drip containing readily utilizable substrates for microbial metabolism. Although the concentrations of carbohydrates (primarily glucose and glycogen) are low in the aqueous phase in comparison to proteins, available concentrations are sufficient to support massive initial microbial proliferation (40,100). After glucose is depleted, microorganisms start using amino acids for energy and as a result produce volatile compounds that are responsible for spoilage odors (31,32,124). Many bacteria, including pseudomonads, produce ammonia during amino acid metabolism, which is a major cause of pH inCopyright 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Table 2 Common Defects of Meats and Causal Bacteria

Defect Slime H2O2 greening H2S greening H2S production Sulfide odor Cabbage odor Potato odor Putrefaction Bone taint Bone taint Pocket taint Internal taint Souring Meat product Meats Meats Vacuum-packaged meats Cured meats Vacuum-packaged meats Bacon Ham Ham Whole meats Bacon Bacon Ham Ham Bacteria Pseudomonas, Lactobacillus, Enterococcus, Weissella, Brochothrix Weisella, Leuconostoc, Enterococcus, Lactobacillus Shewanella Vibrio, Enterobacteriaceae Clostridium, Hafnia Providencia Burkholderia, Pseudomonas Enterobacteriaceae, Proteus Clostridium, Enterococcus Proteus, Vibrio Vibrio, Alcaligenes, Proteus Providencia Lactic acid bacteria, Enterococcus, Micrococcus, Bacillus, Clostridium

Source: Garcia-Lopez et al. (35); Gardner (37); Jay (78).

crease in spoiling meat products (101). Gram-negative bacteria predominate during aerobic spoilage and are generally responsible for the production of putrid and sulfury odors (35). The amino acids cystine, cysteine, and methionine are precursors for hydrogen sulfide, methylsulfide, and dimethylsulfide. Amino acid decarboxylation of lysine yields putrescine. Increasing putrescine levels correlate with increasing pseudomonad counts in meats. Decarboxylation of ornithine or arginine yields cadaverine. Increasing cadaverine levels correlate with increasing Enterobacteriaceae counts (22). It has been noted that pseudomonads preferentially deaminate amino acids, whereas Enterobacteriaceae preferentially decarboxylate (89). Bacteria, other than pseudomonads, responsible for malodorous volatile compounds include Shewanella (Alteromonas) putrefaciens, Proteus, Citrobacter, Hafnia, and Serratia (89). The expression of meat spoilage odors from the degradation of amino acids can be delayed by the addition of glucose to meats. The presence of glucose delays the utilization of amino acids by spoilage bacteria and their subsequent development of sensory spoilage characteristics (4,115). Lactate is another low molecular weight component utilized by meat microflora under both aerobic and anaerobic conditions. Lactate is generally utilized after glucose is depleted and can be used in similar strategies to retard spoilage (116). Indeed, according to Gill (40), as long as readily utilizable low molecular weight substrates are available, meat proteolysis is inhibited. Microbial metabolism in chilled air-stored meats is primarily oxidative (101). Aerobic gram-negative bacteria are the common cause of spoilage of meats stored at 4C, with Pseudomonas species predominating. For example, P. fragi, P. fluorescens, and P. lundensis are the dominant species on beef, lamb, and pork (35). Acinetobacter, Psychrobacter, Moraxella, and psychrotrophic Enterobacteriaceae such as Hafnia alvei, Serratia liquefaciens, and Enterobacter agglomerans also occur but their numbers remain low relative to the dominant pseudomonads. Meats packaged under vacuum or modified atmospheres demonstrate a shift from a diverse flora to one that is predominated by lactic acid bacteria and Brochothrix thermoCopyright 2001 by Marcel Dekker, Inc. All Rights Reserved.

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Figure 2 Hypothetical development of off-odor and slime in meat due to psychrotrophic bacterial growth during storage under various atmospheres at 1C. (Data from Refs. 3 and 95.) sphacta (68). The impact of this shift in microflora is shown in Fig. 2. As illustrated, the time to develop off odors and slime is greatly extended when oxygen is removed from the headspace of packages. Dairy/cheesy odors of meat stored in gas mixtures with carbon dioxide are primarily due to diacetyl, acetoin, and alcohols produced by B. thermosphacta from glucose fermentation (21). End products of B. thermosphacta metabolism differ with the gaseous atmosphere composition. When oxygen tension is low (2 M oxygen), L-lactate, ethanol, and propanol are the main metabolic end products (101). Accordingly, ethanol and propanol could be used as spoilage indicators of meat stored under vacuum of modified atmosphere (98,101). Future prospects of meat substrate and bacterial metabolite studies could point the way to analytical techniques that could assess meat spoilage without resorting to time-consuming microbiological analysis. According to Jay (77), metabolite-based spoilage detection should ensure that (a) the spoilage indicator is not normally present in the food, (b) the indicator concentration should increase with storage time, and (c) the concentrations of the indicator should reflect the most predominant microorganisms and correlate with sensory quality. IV. MICROBIOLOGY OF FERMENTED AND CURED MEATS Comminuted raw meats fermented into various sausages requires the aid of salt, nitrate/nitrite, and desirable fermentative lactic acidproducing bacteria. When properly fermented, pathogens and spoilage bacteria will be eliminated or greatly reduced in numbers, which yields products with superior shelf-life attributes. In addition, desirable sensory properties are achieved. For example, cured red/pink color, robust flavor, and firm texture are unique to this product type (68). Growth and acid production of lactic acid-forming bacteria are promoted by pH below 6.0, water activity (aw) of 0.96 due to salt (2.5 to 3.0%), 100 ppm sodium nitrite, and 0.3% glucose. When stuffed into casings, the redox potential of the sausage is reduced,

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which further enhances activity of the lactic acid bacteria (68). Despite the wide variety of fermented meats around the world and various technical differences during processing, the bacteria that tend to dominate in naturally fermented sausages are usually Lactobacillus sake and Lactobacillus curvatus (61,73,114). Predominance of these two species is based on their ability to grow at reduced aw (0.91) and low temperature (4C). Other psychrotrophic lactics (Carnobacterium spp., Leuconostoc spp. and Weissella spp.) are either less halotolerant (except Weissella halotolerans) or grow poorly below 7C (Lactobacillus pentosus, Lactobacillus plantarum, Pediococcus acidilactici, and Pediococcus pentosaceus) (86). Many traditional fermentations use nitrate as the curing agent. As such, the presence of nitrate-reducing micrococci (Micrococcus varians, Staphylococcus carnosus, Staphylococcus xylosus, or Staphylococcus piscifermentans) is necessary to form nitrite, which is needed for proper quality (color) and safety (antibotulinal activity) (67,68,99). Because the reliability of natural fermentations is occasionally less than desired, most industrial processing of fermented sausages use domesticated starter culture bacteria. These starters are either single or mixed strains of homofermentative lactic acid bacteria. When nitrate is used, micrococci also are included (67,86). Commonly used lactics include L. sake, L. curvatus, L. plantarum, L. pentosus, and P. pentosaceus (P. cerevisiae) (33,66,86,129). Strains are selected primarily for their ability to rapidly acidify, accelerate ripening, and improve color intensity and stability at the fermentation temperature desired (Table 3) (64,65). Spoilage microflora in fresh sausages generally are similar to those found in ground meat. Type of meat, presence of preservatives, and storage temperature and atmosphere will determine the predominant microbes (35). Like fresh meats, products in air-permeable packaging will have pseudomonads predominating during low temperature storage and Enterobacteriaceae during higher temperature storage. Presence of these bacteria on fully cooked products is the result of post-heating contamination, usually during casing removal, slicing, and subsequent handling during packaging. Because of the facultatively anaerobic nature of most Enterobacteriaceae, they tend to predominate in vacuum-and modified atmospherepackaged products stored at high temperatures (10C) (35,104). However, this group competes poorly with lactic acid bacteria under proper chill storage conditions (12). Similarly, gram-negative bacteria usually will not spoil fermented sausages, dried meats, and canned meats (35). Bacteria that are resistant to salt and low aw, nitrite, and fermentation and ripening temperatures will be selected for in fermented sausage products. Many of the same microorganisms are found on cured unfermented products such as hams and bacon. For ex-

Table 3 Lactic Acid Bacteria Used as Starter Cultures for Fermented Sausages at Various Processing Temperatures Bacteria type Thermophiles Mesophiles Psychrotrophs
Source: Holzapfel (68).

Process temperature (C) 3038 2025 1520

Bacterial species Pediococcus acidilactici Pediococcus cerevisiae Lactobacillus pentosus Lactobacillus plantarum Lactobacillus sake Lactobacillus curvatus

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ample, Micrococcaceae, lactic acid bacteria (Carnobacterium and Leuconostoc), Vibrio, Enterobacteriaceae, and other gram-negative bacteria (Psychrobacter, Acinetobacter, and Proteus) are the major microbial groups found on cured products (12,35,37). Among the halophilic (salt loving) vibrios, Vibrio costicola is a common slime former (36). V. MEAT-ASSOCIATED FUNGI Carcasses aged at very low temperatures (5C) can have surface defects caused by molds (Table 1). For example black, white, blue-green, and whisker spots may be evident (25). Cladosporium cladosporioides, Cladosporium herbarum, Penicillium hirsutum, and Aureobasidium pullulans were identified as causative agents of black spot (52,53); Chrysosporium pannorum and Acremonium sp. caused white spots (85). Blue-green spots were associated with Penicillium corylophilum and whisker spots were caused by Thamnidium elegans and Mucor racemosus (85). In addition to bacteria, several molds have been responsible for cured meat spoilage. Low aw and presence of oxygen selects for molds from the genera Aspergillus, Alternaria, Fusarium, Mucor, Rhizopus, Botrytis, and Penicillium (25,78). Some dry-cured products, such as European sausages, Italian salami, and country-cured hams, can support prolific growth of aspergilli and penicillia. There is some speculation that the characteristic flavors of these products are due in part to the presence of these fungi (25,78,105). Like many molds and bacteria, psychrotrophic yeasts are capable of growing on meats during refrigerated storage (Table 1). Most yeast-associated meat spoilage occurs when the product has been treated in such a manner to reduce the level and activity of contaminating bacteria. Such treatments usually include low pH by acidification or low aw by salting, drying, or freezing (25). In fresh meats, however, yeasts generally are unable to compete with bacteria because of their slower growth rates. As such, their numbers remain low in proportion to bacterial counts. Candida spp. are the predominate yeast isolated from raw meats (25,71). Spoilage caused by yeasts is typically related to slime formation on products such as dried sausages, wieners, cured hams, and salami. Debaryomyces spp. and Candida spp. are the predominate yeasts found in processed meats (25). VI. MEAT-ASSOCIATED PARASITES Several microscopic animal parasites may be harbored in meats (Table 4). Parasitic protozoa, flatworms, and roundworms associated with meat animals can be infectious to consumers. Unlike bacteria and fungi, the parasites do not grow in foods but are merely transported either intramuscularly or as surface contaminates. Surface contamination can come from contact with feces or more commonly from use of contaminated water supplies. Fortunately, most parasites are easily killed by proper cooking (80C internal temperature) and handling of products. Long-term freezing (10C for 30 days) or salting of meats also has been shown to inactivate many of the parasites (78). Among protozoa, the coccidian Toxoplasma gondii may cause toxoplasmosis in individuals consuming raw or undercooked meats from cattle, pigs, sheep, and goats (76,121). Sarcocystis hominis and Sarcocystis suihominis may be transmitted by consumption of raw beef and pork (78). Cryptosporidium parvum has been linked to ingestion of leftover beef tripe (78). The flatworm Fasciola hepatica is infrequently found in beef livers. Most other flat-

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Microbiology of Meats Table 4 Parasites Commonly Associated with Meats Parasite Protozoa Genus Toxoplasma Sarcocystis Cryptosporidium Fasciola Taenia Trichinella Meat Beef, pork, sheep, goat Beef Beef Beef liver Beef, pork Pork

161

Flatworms Tapeworms Roundworms

Source: Jay (78).

worms are distributed in fish rather than meat animals (78). The common tapeworms Taenia saginata (Taeniarhynchus saginatus) and Taenia solium can cause mild human illness in consumers eating raw or undercooked beef and pork, respectively. Of more severity is the illness trichinosis, caused by consumption of raw or undercooked pork containing the roundworm Trichinella spiralis. This illness can occasionally be fatal (78). VII. MICROBIAL MODELING

Predictive modeling of microbial growth and survival in meats has become an increasingly important tool in studying the behavior of spoilage and pathogenic microorganisms under different environmental conditions. Predicting the impact of intrinsic (nutrients, pH, salt, nitrite, etc.) and extrinsic (atmosphere, temperature) factors on microorganisms can help processors and regulators determine optimum conditions needed for enhanced quality and safety. Two computer-based modeling programs are available: Food Micromodel (Leatherhead, Surrey, UK) and Pathogen Modeling Program (USDA, Wyndmoor, PA). Example outputs from the USDA model are illustrated in Figs. 3 through 5. Figure 3 shows predicted growth potential of E. coli O157:H7 under specified atmosphere, temperature, pH, salt, and nitrite conditions. Figure 4 demonstrates the predicted amount of time needed to achieve a 3 log or greater reduction in the numbers of L. monocytogenes at pH 3.5, 16.9C, and 0.5% NaCl. Figure 5 illustrates the predicted survival of S. typhimurium at 0C with increasing gamma irradiation doses. VIII. SUMMARY The numbers and types of microorganisms found on meats are determined by the environment under which the animals were raised and processed, and the meat packaged and stored. With current technology it is nearly impossible to produce sterile meats without excessive thermal or irradiation processing. That said, proper animal husbandry, workplace sanitation, and processing will produce edible meats with acceptable microbial numbers and low or no human pathogens. However, it must be expected that raw meats will contain potential human pathogens. In addition, with the possible exception of canned meats, most processed meats will have a limited shelf life that is dictated by the quantity and kinds of spoilage microorganisms on the product. Meat processors have the expectation that farmers and feedlots will provide animals for slaughter that are safe for meat production. Like-

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Figure 3 Predicted aerobic growth of Escherichia coli O157:H7 using the USDA Pathogen Modeling Program.

Figure 4 Predicted low pH inactivation time needed for Listeria monocytogenes using the USDA
Pathogen Modeling Program.

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Microbiology of Meats

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Figure 5 Predicted irradiation inactivation curve for Salmonella typhimurium using the USDA Pathogen Modeling Program.

wise, consumers have the expectation that meat products available for consumption are safe and wholesome. Failure by the meat industry to meet these expectations frequently leads to microbial problems and ultimately decreased consumer demand. Therefore, understanding the quantity and nature of meatborne microorganisms remains a critical issue for long-term viability of the industry. ACKNOWLEDGMENTS The USDA Pathogen Modeling Program ver. 5.1 was developed by R. L. Buchanan, Ph.D., R. C. Whiting, Ph.D., and A. R. Pickard, Ph.D., at the Microbial Food Safety Research Unit of the USDA/ARS Eastern Regional Research Center in Wyndmoor, PA. REFERENCES
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